1. Field of the Invention
It is frequently desirable to be able to bind, detect, or remove a particular compound in a medium suspected or known to contain that compound. The present invention relates to conjugate monomers containing binding-pair members, to polymers synthesized from these conjugate monomers and to the de novo synthesis of these polymers. The polymers contain the binding-pair members as integral parts of their structure and find use in selective removal of binding-pair-member cognates from solution as well as in various immunoassay procedures and immunization regimes.
2. Description of the Relevant Art
Polymerization of monomers or oligomers to form larger polymeric molecular structures (polymers) or the initiation of copolymerization of monomers with polymerizable polyunsaturated compounds is fundamental to polymer chemistry. Polymers may be formed from a single monomeric species (homopolymers), from a mixture of different monomers (copolymers), from polymerizable polyunsaturated compounds containing olefinic or acetylenic unsaturation, or from a mixture of polymerizable polyunsaturated compounds and one or more monomers. Linear, branched or cross-linked polymeric structures are possible.
By varying the chemical composition and/or ratios of the monomer and/or polyunsaturated compound, it is possible to form either water-soluble or water-insoluble polymers which have a broad range of chemical and physical properties. For example, water-soluble monomers (such as acrylamide) may be homopolymerized to form water-soluble homopolymers. Such monomers may also be copolymerized with less water-soluble monomers (such as N-alkyl or N,N-dialkyl acrylamide) or with cross-linking monomers (such as N,N'-methylenebisacrylamide) to form water-insoluble copolymers. Some water-soluble monomers (such as 2-hydroxyethylmethacrylate or acrylonitrile) may be homopolymerized to form water-insoluble homopolymers.
Water-insoluble polymers (such as polysaccharides and polyacrylics) have been commonly used in the fields of biochemistry and immunology (affinity chromatography and immunoassay) as solid phase supports with passively adsorbed or covalently linked antibodies. To date, however, antibodies have only been immobilized on preformed insoluble polymeric materials. For example, antibodies can be covalently bonded to cyanogen bromide-activated beads of Sepharose-4B or beads of cross-linked acrylic polymers (U.S. Pat. No. 3,957,741). Also, see Affinity Chromatography and Related Techniques, Proceedings of the Fourth International Symposium, Veldhove, The Netherlands, June 22-26, 1981, Ed. P. C. J. Gribnau, J. Visser and R. C. S. Nivard, Elsevier Scientific Publishing Company, New York, 1982. The immobilized antibodies can then be used to specifically bind antigens to the solid surface of the beads followed by extensive washing to remove other adsorbed substances. Subsequently the bound antigens can be eluted from the antibody/polymer mat by treatment with chaotropic agents, high salt concentrations or low pH buffers. Antibodies have also been confined within encapsulated membranes for use in affinity chromatography (U.S. Pat. No. 4,257,884).
U.S. Pat. Nos. 3,314,905 and 3,453,222 disclose reaction products of proteinaceous materials and certain esters to form substances which are capable of polymerization (modified proteins). The reaction conditions under which these modified proteins are synthesized are harsh, e.g., elevated temperatures (typically 50.degree. C.) and high pH (typically &gt;8). The end products have use as resin-protein wood adhesives or flocculating agents.
U.S. Pat. No. 2,548,520 discloses high molecular weight materials prepared by copolymerizing proteins having unsaturated radicals chemically united therewith with unsaturated polymerizable monomers or their partial polymerization products. Production of these high molecular weight materials generally requires temperatures up to 100.degree. C. Such high temperatures are not well tolerated by most proteins. Thus methods as described are unsuitable for producing polymers of biologically active molecules.
U.S. Pat. No. 3,969,287 discloses a method for the preparation of carrier-bound proteins wherein the protein is reacted with a coupling compound containing at least one double bond capable of copolymerization. The carrier is provided as a water-insoluble solid or is produced in situ by the polymerization of water-soluble monomers in the presence of the protein/coupling compound adduct. The proteins utilized in the methods of this invention are typically enzymes, and, of those disclosed, none contain multiple subunits.
In immunoassays (see Campbell, D. H. and Weliky, N. Methods in Immunology and Immunochemistry, Ed. Williams and Chase, Vol. I, Academic Press, New York, 1967), antibodies or antigens have been passively adsorbed to surfaces, e.g., to the wells of microtiter plates, plastic beads (U.S. Pat. No. 4,225,784), or latex particles. The solid phase antibody/polymer matrix provides a selective binding surface which, following antigen binding, can be washed to separate bound from unbound reactants. Also known is (a) the covalent bonding of antigens or antibodies to latex beads (U.S. Pat. No. 4,181,636) or high-refractive-index particles (U.S. Pat. No. 4,401,765) to measure agglutination reactions or (b) the binding of antibodies to fluorescent polymer beads to provide specific tags for cell-surface antigens (U.S. Pat. No. 4,166,105).
While the insoluble polymer/antibody materials described above provide a surface upon which selective biochemical or immunological reactions can occur, the polymers formed by bonding a molecule to an already formed polymeric material are limited in that the spacing, steric accessibility, and number of antibody molecules bound per unit length of polymer cannot be precisely or reproducibly controlled. Lot-to-lot variation is commonly encountered during the manufacture of such solid phase polymers. In certain end-use applications where reproducibility and standardization are important (e.g., immunoassays), the variation in composition of the solid phase polymer/antibody material may not be acceptable.